Botulinum Neurotoxins (BoNTs) are the most toxic substances known and classified as Category A biothreat agents. BoNTs are zinc metalloproteases that cleave and inactivate proteins involved in synaptic vesicle fusion and acetylcholine release. Therapeutic applications of botulinum toxins have increased steadily over the years requiring new and reliable assays to assess the potency and stability of BoNT products. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for a revolutionary quantitative, ultrasensitive and -specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with exquisite single-well multiplexing potential, minimal sample requirements, and extremely simplified handling procedures (no separation/washing steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby two (or more) target recognition molecules are tagged with different color fluorescence dyes. Coincident confocal detection of two (or more) colors constitutes a positive target detection event, allowing identification of biomolecules in solution and detection of numerous targets simultaneously. NTI recently achieved expansion from 2-color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power. Furthermore, recent work by our consultants Profs. Stephen Quake and Shimon Weiss demonstrates successful merging of microfluidics-based sample handling with 2c-ALEX spectroscopy for quantification of enzymatic activity, a new breakthrough approach for assay miniaturization and increased throughput termed single molecule optofluidics. NTI's long-term goal is to develop rapid, highly multiplexed (with a capacity of >100 analytes per sample), ultrasensitive and -specific, quantitative, cost-effective, and fully automated, protein- and nucleic acid-based syndrome-driven tests that require minimal patient samples. In this Phase I SBIR application, we propose to adapt ALEX-based single molecule optofluidics for development of an innovative, completely solution-based in vitro endopeptidase assay, overcoming limitations of animal- and cell-based assays, to determine BoNT/A potency and stability based on quantifying its protease activity by measuring fluorophore-labeled substrate cleavage. Specific Aims are: 1. Substrate peptide synthesis and fluorophore/quencher conjugation. 2. BoNT/A detection and quantification using ALEX-based single molecule optofluidics. 3. Determination of specificity, sensitivity (d100 fM), linear range of quantification (e4 log orders), & time (1 hr). Phase II will be dedicated to assay optimization, automation, prototype development, and, if considered desirable, expansion to include other BoNT substrates for rapid multiplexed (single-well) discrimination and quantification. Software will be developed suitable for user-friendly operation of a fully integrated ALEX-based testing system for routine commercial release testing of BoNT products. It may serve as basis for future development of an FDA-approved diagnostic system for BoNT and other infectious diseases agents testing. PUBLIC HEALTH RELEVANCE: Nesher Technologies, Inc. intends to develop an innovative alternative approach, based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, to the currently used mouse intraperitoneal injection assay which measures activity in Mouse Units (MU), with 1 MU of activity defined as the LD50 dose (death within 72 to 96 hours in 50% of the mice), in order to allow for standardization of potency units for Botulinum Neurotoxin products made by different manufacturers. This will greatly reduce use of animals and will also complement Nesher Technologies' federally-funded efforts of instrument and reagent development for tests for bioterror agents, infectious and genetic diseases, and early cancer detection, thereby translating cutting-edge innovations in nanobiotechnology into benefits for the society at large by saving human lives, monitoring the population for bioterror attacks and disease outbreaks, and reducing healthcare costs.